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Predication of the Underlying Protective Effect ofSaffron on Parkinson's Disease via NetworkPharmacology and Molecular DockingJunjie Gong 

Zhejiang University of TechnologyZijin Xu 

Zhejiang University of TechnologyShushu Hao 

Nanjing Medical UniversityBoqian Chen 

Nanjing Medical UniversityShengcheng Zhuang 

Nanjing Medical Universityguojun Jiang 

Zhejiang Xiaoshan HospitalSZ Bathaie 

Tarbiat Modares Universityping wang  ( wangping45@aliyun.com )

Zhejiang University of Technology https://orcid.org/0000-0001-5720-8687

Research

Keywords: Network pharmacology, Molecular docking, Saffron, crocin, Mechanism, Parkinson's disease

Posted Date: January 13th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-143357/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

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AbstractBackground

Reports indicate that saffron originated from Iran then introduced into mainland China through the earlierestablished trans-Tibet trade routes in ancient China. Based to the theory of traditional Chinese medicine,saffron functions by promoting blood circulation and removing blood stasis, cooling blood anddetoxi�cation, as well as relieving depression for tranquilization. Modern medical research hasdemonstrated several properties of saffron including antitumor, antidepressant, enhancement ofimmunity, cardioprotection. Notably, recent studies introduce that saffron has a neuroprotective effect,speci�cally in the treatment of Parkinson's disease in recent years. Nevertheless, the underlying molecularmechanism remains elusive so far. As such, this study aims to predict the underlying mechanismof saffron on Parkinson's disease through network pharmacology and molecular docking.

Methods

A functional-based network pharmacology and molecular docking model was constructed for dissectingthe underlying mechanism of saffron on Parkinson's disease. Based on the Traditional Chinese MedicineSystem Pharmacology database (TCMSP) and the literature reviews, the putative targets of Saffron werecollected. Further, the interaction networks of Drug- candidate compounds targets-Therapeutic targets-Disease were mined using the Cytoscape software. Gene Ontology (GO) and Kyoto Encyclopediaof Genes and Genomes (KEGG) pathway enrichment analyses were conducted and the String databasewas used to analyze the protein interaction network. Finally, Molecular docking was used to predict theinteraction between the components in Saffron and key targets by MOE 2014.09 software.

Results

A total of 9 candidate compounds from saffron corresponding to 52 therapeutic targets of Parkinson'sdisease were identi�ed. The neuroprotective effect of saffron in the treatment of PD was attributed to thestrong binding between crocin (including crocin I and crocin II) as well as key targets including CASP3,IL6, and MAPK8. Additionally, the auxiliary neuroprotective effects might have originated from quercetin,kaempferol, isorhamnetin, crocetin, safranal, and picrocrocin because of their slightly weaker bindingcapacity to the key targets.

Conclusion

Saffron exhibits a synergistic effect via multiple targets and pathways in the treatment of Parkinson'sdisease and our systemic pharmacological analysis provides a basis for the clinical application and in-depth study of saffron.

Introduction

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Parkinson's disease (PD) is a neurological disorder characterized by degeneration of dopaminergicneurons in the substantia nigra pars compacta, with dyskinesia as the prominent clinical symptom whichincludes resting tremor, bradykinesia, rigidity, and postural instability [1]. The pathological mechanism ofPD is complex, and multiple biological mechanisms are related to PD, including oxidative stress, immunein�ammation, mitochondrial dysfunction, and accumulation of toxic proteins [2-6]. Additionally,epidemiological studies have suggested that several factors play key roles in the development of PD,including, race, age, environment, lifestyle, and heredity among others [7-10]. Notebly, a retrospectiveanalysis of PD in 2016 revealed that the number of patients nearly tripled from 2.5 million in 1990 to 6.1million across the globe [11]. In China, the current situation remains unoptimistic due to the surge of theaging population. Besides, a statistical analysis estimates that the Chinese PD patients will reach 4.94million, more than half the global incidence by 2030 [12]. Dopamine-related drugs including levodopa arepresently used as �rst-line therapy in clinical treatment based on the authoritative guideline [13, 14]. As aconsequence, although it can alleviate symptoms in the short term, there is still lack of evidence-basedmedical evidence that the drug can reverse the pathological process of PD. The problem that has to bepaid attention to in the process of clinical treatment is that patients receiving long-term treatment willdevelop tolerance, so it is necessary to continuously increase the dose to achieve the therapeutic effect[15-17]. The use of such drugs has caused many side effects, including dyskinesias and behavioralinhibition and control damage. Therefore, continuing to carry out in-depth research on the mechanism ofPD and discovering better therapeutic drugs is the direction of future efforts [18].

Crocus sativus L., commonly known as saffron, is a perennial stemless herb of the Iridaceae. Notably,commercial saffron comprises the dried red stigma. Saffron is widely cultivated in Europe, Turkey, Iran,Central Asia, India, China, and Algeria, of which Iran has been the world's major producer and exportersince time immemorial (85% of the global saffron production) [19]. A lot of Islamic traditional medicineliterature introduced saffron as an astringent, resolvent, and concoctive drug with therapeutic effects ongastro-hepatoprotective, urogenital disorders, antidepressants, ocular disorders, etc. [20]. Theaccumulating pharmacological studies of saffron indicated that it can elicit anti-tumor, anti-in�ammatory,antioxidant, immunomodulatory effects, and neuroprotective action [21-25]. Notably, the pharmacologicalmechanism of anti-oxidation, anti-in�ammatory and inhibition of toxic α-Syn protein aggregation ofsaffron extract may be related to ameliorating the symptoms of PD patients [26-28]. The use of newresearch methods to clarify the precise pharmacological mechanism of saffron has important clinicalsigni�cance for further promoting the use of the drug in the treatment of PD patients. Scientists believethat the active substance groups of traditional Chinese medicine (TCM) play a therapeutic role via multi-target and multi-pathway. Of note, network pharmacology is an effective method to understand humanbody-drug interaction from a macro perspective, and to further explore the scienti�c problem onmechanism by which the complex components of TCM interfere with disease targets [29]. Given that theprecise mechanism of saffron in the treatment of PD remains unclear, we sought to predict the underlyingmechanism of saffron on PD through network pharmacology and molecular docking. First, we attemptedto screen the related active component of saffron in therapeutic target on PD and further conductedinterrelation analysis using multiple databases. Moreover, to clarify the mechanism of saffron in the

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treatment of PD, the bond strength between components and disease targets were evaluated bymolecular docking. The �owchart of the experimental procedures is shown in Fig. 1.

MethodsScreening the candidate compounds and targets of saffron

Conventionally, oral administration of Saffron was usually the selected route, hence absorption,distribution, metabolism, and excretion (ADME) regulated the process of drug exerting its action. Oralbioavailability (OB) and drug-likeness (DL) have been introduced to characterize the pharmacokineticcharacteristics of active components, and to further provide criteria for screening active componentsfrom TCM [30, 31]. OB is a vital index to describe the relative amount of drug absorption into the bloodcirculation, whereas DL refers to the similarities between the active components and most currentlyknown drugs in physical and chemical properties. Generally, compounds that meet these basicpharmacokinetic parameters might accelerate new drug discovery and improve the success rate of drugdevelopment. Based on the candidate compounds screening thresholds, this study set OB≥30% andDL≥0.18 to screen the active components of saffron using the traditional Chinese medicine systempharmacology database and analysis platform (TCMSP, http://lsp.nwu.edu.cn/tcmsp.php) [32, 33]. Assupplementary, a literature search was conducted in PubMed (https://pubmed.ncbi.nlm.nih.gov ), usingthe items “Parkinson”, “Crocus sativus L”, and “Saffron” as keywords. Based on this, we added thefollowing active components: Crocin I, Crocin II, Safranal, and Picrocrocin. Despite these 4 componentsnot meeting the above requirements, it was clearly mentioned highlighted in relevant literature forneuroprotective and the therapeutic effect of PD [34-37]. Further, the biological targets of the candidatecompounds in Saffron were gathered from TCMSP. Moreover, α-Synuclein protein (α-Syn, protein-codinggene known as SNCA) was supplemented into the �nal result because of it being the main components inthe Lewy body, the most important pathological markers of PD. It has been convincingly demonstratedthat Crocin I and Crocin II could interact with α-Syn and inhibit the formation of aggregates in vitro andcomputer simulation studies [28, 38]. The last step was to delete duplicate targets and further ensuredata quality as well as standardization using the Uniprot database (https://www.uniprot.org/).

Collection of the con�rmed and potential therapeutic targets of PD

Notably, disease therapeutic targets usually include receptors, enzymes, ion channels, transporters etc.The interaction between drugs and targets triggered changes in physiological and biochemical functionsof the body, to realize the regulation of drugs on the pathological and physiological state of the body andachieve the purpose of disease treatment [39, 40]. In light of all of this, the con�rmation of diseasetargets is an important basic work in drug development and clinical diagnosis and treatment. Searchterms including“Parkinson’s disease” as keywords was used to conduct a systematic data search fordisease targets from databases with no date restrictions, including, Online Mendelian Inheritance in ManDatabases (OMIM, http://www.omim.org/), Genecards Databases (http://www.genecards.org/, cut offvalue larger than 20), DrugBank Databases (https://www.drugbank.ca/), Comparative Toxicogenomics

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Database (CTD, http://ctdbase.org/, Score greater than 40). This selection of databases was based onsusceptibility genes, con�rmed drug targets, and environmental exposure factors hinged on the focus ofdifferent databases. Further, the search results were comprehensively analyzed and converted to thecorresponding o�cial gene symbol by Uniprot Database to �nally obtain the de�nitive targets of PD.

Construction of the drug-candidate compounds -therapeutic targets-disease network

Further interrelation analysis was performed to obtain the intersection of candidate compounds targetsand therapeutic targets all the above collected, namely common targets, these candidate compoundsfrom saffron might act directly on the therapeutic targets of PD. Then, the network of drug-candidatecompounds-therapeutic targets-disease was constructed using Cytoscape software version3.7.2. Nodeswith different shapes and colors represented different properties of the nodes in the visual networkdiagram. Speci�cally, the connecting line represented the subordination relationship of drug-compoundsor disease-targets and the compound-targets action relationship between nodes [41].

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichmentanalysis

In this work, we took a step further transforming the o�cial gene symbol of common targets into entrezIDusing the “org.Hs.eg.db” R package of R software version 3.6.2 (x64). Then, Gene Ontology (GO) andKyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed usingthe “clusterPro�ler” R package, meanwhile, associated signaling pathways were rendered using the“Pathview” R package. The results were presented in the form of a bar chart and a bubble chart. P-value <0.05 or Q value < 0.05 were considered statistically signi�cant enrichment.

Construction of the protein-protein interaction (PPI) network for the therapeutic targets of PD

To predict all the potential interactions of common targets between candidate compounds targets andtherapeutic targets, all the common targets were inputted into the String database (https://string-db.org/cgi/input.pl), with the species limited to “Homo sapiens” and “minimum required interactionscore>0.4” as the basis of con�dence for the interaction between proteins. Subsequently, the basictopological parameters of protein-protein interaction network including the degree value of the node(Degree), closeness centrality (CC), and betweenness centrality (BC) were calculated using the NetworkAnalyzer tool function of String database, all the results were presented as a bar chart and 3D scatterplot.

Molecular docking simulation

The concept of molecular docking refers to placing small molecular ligands in the binding region ofmacromolecular receptors through computer simulation then calculating physical and chemicalparameters to predict the binding a�nity between them. Brie�y, the X-ray crystal structure of the targetprotein was downloaded from the protein data bank database (PDB, http://www.rcsb.org/), and themolecular docking was performed using MOE 2014.09 software. The Triangular Matching docking

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method was used to dock the molecules, then, 30 conformations of each ligand-protein complex weregenerated based on docking score. The docking poses between the active components and the bindingpocket residues of the target protein were evaluated via visual inspection relying on the visualizationfunction of the software. Finally, the clear images of ligand-protein interactions were retained andoutputted

ResultsCandidate compounds and targets of Saffron

A total of 9 candidate compounds were collected, 4 of which were collected from the primary literature asmentioned above, whereas the remaining 5 including Quercetin, Kaempferol, Isorhamnetin, Crocetin, n-Heptanal were screened from the TCMSP database. Also, the TCMSP database was used for targetanalysis of candidate compounds, on this basis, the repeated targets were consolidated and the above-mentioned con�rmed target SNCA was added. A total of 92 targets of candidate compounds werecollected and the basic pieces of information, including OB, DL, and numbers of corresponding targetsare listed in Table 1.

Table 1 The list of 9 candidate compounds of Saffron and their basic pieces of information

ID compound OB % DL Number of target

MOL000098 Quercetin 46.43 0.28 45

MOL000422 Kaempferol 41.88 0.24 21

MOL000354 Isorhamnetin 49.60 0.31 8

MOL001406 Crocetin 35.30 0.26 7

MOL001389 n-Heptanal 79.74 0.59 2

MOL001405 Crocin I 2.54 0.12 1

MOL001407 Crocin II 1.65 0.21 1

MOL000720 Safranal 39.56 0.04 5

MOL001409 Picrocrocin 33.71 0.04 2

Potential therapeutic targets of PD

Results from four databases are merged and de-duplicated to provide a list of all con�rmed and potentialtherapeutic targets of PD. A total of 38 therapeutic targets were screened out from Drugbank. The resultswere more convincing because the resources provided by Drugbank came from the con�rmationinformation of listed drugs. Up to 104 PD related targets with cut off value larger than 20 were collectedfrom the Genecards database while 29 therapeutic targets were collected from the OMIM database.

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Notably, the Genecards database provides genes related to common human diseases, while the OMIMdatabase focuses on the relationship between disease phenotypes and their pathogenic genes. CTDdatabase considered the genes affected by PD related environmental toxicant exposure, and a total of466 therapeutic targets were collected when the screening score was set as greater than 40. A total of556 PD therapeutic targets were collected from different databases, as shown in Fig. 2. There was notmuch overlap between different databases, indicating that the therapeutic targets collected in this studywere quite complete.

Common targets of between compounds targets of saffron and therapeutic targets of PD

Further analysis revealed that 52 common targets were observed in both candidate compounds targets ofsaffron and therapeutic targets of PD (Fig. 3) and their basic information regarding Uniprot ID, o�cialgene symbol of target, and protein full name of target (Table 2). Then, this result was classi�ed based onthe main molecular function of the target proteins, and these molecular functions concentrated onapoptosis, oxidative stress, in�ammation, cholinergic system disorder, and toxic protein damage. Thiswas consistent with the recent studies on the pathological mechanism of PD, (Fig. 4).

Table 2. Basic information of common targets

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Uniprot ID Target gene Target protein

P37840 SNCA Alpha-synuclein

P09488 GSTM1 Glutathione S-transferase Mu 1

P05231 IL6 Interleukin-6

P15559 NQO1 NAD(P)H dehydrogenase

P09211 GSTP1 Glutathione S-transferase P

P10415 BCL2 Apoptosis regulator Bcl-2

Q16236 NFE2L2 Nuclear factor erythroid 2-related factor 2

P42574 CASP3 Caspase-3

P01100 FOS Proto-oncogene c-Fos

P22303 ACHE Acetylcholinesterase

P15692 VEGFA Vascular endothelial growth factor A

Q04206 RELA Transcription factor p65

P55211 CASP9 Caspase-9

P45983 MAPK8 Mitogen-activated protein kinase 8

P37231 PPARG Peroxisome proliferator-activated receptor gamma

P03372 ESR1 Estrogen receptor

P24385 CCND1 G1/S-speci�c cyclin-D1

P49841 GSK3B Glycogen synthase kinase-3 beta

P25963 NFKBIA NF-kappa-B inhibitor alpha

P08684 CYP3A4 Cytochrome P450 3A4

P29474 NOS3 Nitric oxide synthase, endothelial

P09874 PARP1 Poly [ADP-ribose] polymerase 1

P04798 CYP1A1 Cytochrome P450 1A1

Q16665 HIF1A Hypoxia-inducible factor 1-alpha

P04792 HSPB1 Heat shock protein beta-1

P05362 ICAM1 Intercellular adhesion molecule 1

P01106 MYC Myc proto-oncogene protein

P27169 PON1 Serum paraoxonase/arylesterase 1

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P10275 AR Androgen receptor

P00533 EGFR Epidermal growth factor receptor

P14635 CCNB1 G2/mitotic-speci�c cyclin-B1

P23219 PTGS1 Prostaglandin G/H synthase 1

P19320 VCAM1 Vascular cell adhesion protein 1

P17252 PRKCA Protein kinase C alpha type

P02741 CRP C-reactive protein

P28161 GSTM2 Glutathione S-transferase Mu 2

O15392 BIRC5 Baculoviral IAP repeat-containing protein 5

Q14790 CASP8 Caspase-8

Q03135 CAV1 Caveolin-1

P16435 POR NADPH--cytochrome P450 reductase

P08172 CHRM2 Muscarinic acetylcholine receptor M2

Q13085 ACACA Acetyl-CoA carboxylase 1

P04049 RAF1 RAF proto-oncogene serine/threonine-protein kinase

P07339 CTSD Cathepsin D

Q16678 CYP1B1 Cytochrome P450 1B1

P14867 GABRA1 Gamma-aminobutyric acid receptor subunit alpha-1

P35869 AHR Aryl hydrocarbon receptor

P06400 RB1 Retinoblastoma-associated protein

P11229 CHRM1 Muscarinic acetylcholine receptor M1

P19419 ELK1 ETS domain-containing protein Elk-1

P35348 ADRA1A Alpha-1A adrenergic receptor

P20309 CHRM3 Muscarinic acetylcholine receptor M3

Construction of the drug- candidate compounds-therapeutic targets-disease network

The network of the drug, candidate compounds, therapeutic targets, and PD was built to analyze thecomplex interactions between saffron and PD. As shown in Fig.5, the network comprised 153 edges and63 nodes including 52 therapeutic targets of PD represented by green circular nodes, and 9 compoundsof saffron represented by orange triangle nodes. The node size and depth of the color determined thedegree value of the node, for further explanation, the larger the size and the darker the color, the greater

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the degree value of the node. The current �ndings con�rm that the relationship between candidatecomponents and therapeutic targets is not a simple rule like one-to-one correspondence, but complex, forinstance, one-to-many or many-to-one. Many candidate components were related to multiple therapeutictargets of PD, particularly quercetin and kaempferol, which might be the important compounds in thetreatment of PD.  

GO and KEGG enrichment analyses

The purpose of GO enrichment was to clarify the potential mechanism of saffron in the treatment of PDfrom the biological process (BP), cellular component (CC), molecular function (MF), respectively. Theresults of GO enrichment of 52 common targets were depicted as a representative bar chart, the longerthe bar, the smaller the p-value is, namely, the more signi�cant the enrichment is. As shown in Fig. 6, thetop 6 biological processes implicated in the treatment of PD were cellular response to chemical stress,response to the xenobiotic stimulus, cellular response to the xenobiotic stimulus, response to the metalion, response to oxidative stress, and cellular response to oxidative stress. In terms of cellularcomponents, the top 6 were membrane raft, membrane microdomain, membrane region, transcriptionregulator complex, an integral component of postsynaptic membrane, and axon terminus. Additionally,DNA-bind transcription factor binding, RNA polymerase II-speci�c DNA-binding transcription factorbinding, activating transcription factor binding, ubiquitin-like protein ligase binding, and glutathionebinding were the top 6 molecular functions implicated in the treatment of PD. Fig. 7 is a simpli�ed bubblechart that describes the results of KEGG enrichment, as its purpose was to clarify the top 16 signalpathways for the treatment of PD using saffron. In general, PD-related pathways including PI3K/Aktsignaling pathway, apoptosis signaling pathway, and TNF signaling pathway showed signi�cantenrichment. For clearer presentation, the speci�c position and function of common targets are colored redin the signaling pathway in Fig .8.

Protein-protein interaction (PPI) network for the therapeutic targets of PD

In this work, the protein-protein interaction (PPI) complexity of 52 common targets is clearly shown in Fig.9 (A), Further, the degree values of the top 30 proteins in interaction relationship are shown in Fig. 9 (B).Here, the degree value of protein was a critical parameter, a direct indication of the importance in thenetwork, and the protein with high a degree value was more likely to be the core target in the treatment ofPD. To describe the topological properties of the nodes and comprehensively evaluate the status ofproteins in the PPI network, a three-dimensional scatter plot was used to show the degree value,closeness centrality (CC), and betweenness centrality (BC) on each node (Fig. 9) (C). There was asigni�cant positive correlation between them. Generally, the closeness centrality and betweennesscentrality of protein with high degree value was also greater, indicating that the protein directly interactswith many proteins and act as a "bridge" to mediate the indirect interaction between other proteins. Thisconclusion implies that CASP3, IL6, MAPK8 were the three most important genes in the PPI network,which might be the core proteins of PD.

Molecular docking

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Multiple studies have reported that the active components of saffron, including quercetin, kaempferol,isorhamnetin, crocetin, crocin I, crocin II, safranal, and picrocrocin exhibit anti-apoptotic and anti-in�ammatory properties [42-48]. The above results suggest that CASP3, IL6, and MAPK8 are at the core ofthe PPI network, consistent with the literature, there were closely related to neuronal apoptosis andneuroin�ammation of PD [49-51]. Therefore, additional extensive research is essential to explore theeffect of saffron on the therapeutic targets of PD. The molecular docking technology used in this workcan be presented as macro, micro, and local state of the interaction between compounds and coreproteins. The crystal structure of CASP3 (PDB: 5IC4), IL6 (PDB: 1N26), and MAPK8 (PDB: 4QTD) wereretrieved from the PDB database and executed protonated and energy minimized before moleculardocking. The docking score of the 8 compounds and 3 proteins was shown by the heatmap (Fig. 10). Thescores were used to describe the binding free energy between the compounds and the proteins, i.e, thesmaller the value, the lower the binding free energy, indicating a more stable binding between compoundsand the proteins. Furthermore, it was observed that the binding free energy of crocin I and crocin II wasless than -8.0 kJ/mol. It was also suggested that crocin I and crocin II selectively act on the targetsassociated with apoptosis and in�ammation, which then functioned as the limitation of in�ammationand apoptosis to play a neuroprotective role.

DiscussionBeing a complex disease affecting the central nervous system, PD has become a major threat to humanhealth, and, researchers indicate that the situation is getting worse in the years ahead. The apparentpathological mechanism is a decrease of dopaminergic neurons in the substantia nigra pars compacta(SNpc), causing the depletion of dopaminergic neurotransmitters in the striatal projection neurons, andeventually leading to the functional imbalance between the dopaminergic system of substantia nigra andthe cholinergic system of the striatum [52]. The death of dopaminergic neurons involves complexpathogenic factors and pathological mechanisms, suggesting that it is di�cult for drugs with a singletarget to achieve a satisfactory therapeutic effect in the treatment of PD. Traditional TCM has a longapplication history in China and other Asian countries, besides, TCM is considered the pioneer of “multi-component and multi-target pharmacology” [53]. Based on the theory of TCM, PD belongs to the categoryof tremble syndrome which considers de�ciency of body caused by old age, liver and kidney de�ciency,and wind evil stirring internally caused by tendons dystrophy as its etiology. According to the theory ofsyndrome differentiation and treatment (“bian zheng shi zhi” in Chinese) in traditional Chinese medicine,the treatment principle of PD is to tonify liver and kidney, balance the liver and extinguish wind, nourishblood and soften tendons. Notably, this work was intrigued by a classical famous formula, Five DragonTremor Decoction (“wu long zhen chan tang” in Chinese) that was �rst documented in the classicalChinese medical book Discussion on Febrile Disease (“wen bing Tiao bian” in Chinese) in the QingDynasty (18th century). Saffron is an important adjuvant in this formula and plays a signi�cant role inpromoting blood circulation and removing blood stasis. Further, it has been suggested that saffron mightbe a potential drug for the treatment of PD, a scenario that warrants in-depth investigation. Therefore,TMC has its understanding and characteristics on the pathological mechanism and treatment of PD.

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Summarily, it executes a multi-component, multi-target, and multi-signaling pathways to achieve thepurpose of disease treatment. Here, we used the network pharmacology method to clarify how thecomponents of saffron interfere with targets and related pathways of PD disease.

Recent research �ndings reveal that saffron extract harbors an antidepressant effect by inhibiting centralin�ammation, ameliorating oxidative stress, increasing the expression of synaptic plasticity-relatedproteins in hippocampal neurons, and regulating intestinal micro�ora [54-56]. Additionally, our �ndingsshow that crocin reduced the structural damage in soma volume and axon length of neurons andinhibited their spontaneous discharge in dopaminergic neurons in the ventral tegmental area (VTA) [57].Moreover, evidence from clinical trials con�rms the antidepressant e�cacy of saffron extract comparableto Citalopram [58]. The above research results inspire the exploration of whether saffron extract protectsthe substantia nigra pars compacta, which is close to the space of VTA and dominated by dopaminergicneurons. So far, no clinical trial data supports the idea that saffron is a potential drug in preventing andtreating PD, however, the biological activity of neuroprotective is gradually being clari�ed in vitro studies.In an animal model of PD induced by rotenone, an inhibitor of the Mitochondrial Electron Transport Chain(ETC), crocin activated the PI3K/Akt pathway, then, phosphorylated Akt inhibited the phosphorylation ofGSK-3β to reduce the phosphorylation of α-Syn and eventually inhibited the aggregation of α-Syn [59].Importantly, phosphorylated α-Syn is more aggressive and more toxic compared to its prototypes [60].Also, the activation of mTOR by p-Akt promoted the expression of downstream effector p70S6K andincreased the level of Bcl-2 in preventing cell apoptosis. Also, p-Akt inhibited the expression of Caspase-9by inhibiting the transcription of FoxO3a to prevent the initiation of the apoptosis cascade reaction [59].Therefore, the activation of the PI3K/Akt pathway is an effective intervention approach in reducing theabnormal aggregation of toxic proteins and preventing cell apoptosis. PI3K/Akt signal transductionpathway was signi�cantly enriched in our study, and this was consistent with a growing body of evidencein the literature.

Additional studies have recently suggested central in�ammation as one of the key factors causing thedeath of dopaminergic neurons. In recent years, there has been an increasing focus on PD-relatedneuroin�ammation caused by the aggregation of α-Syn and microglial activation caused by intestinal�ora imbalance [61]. Also, crocin and crocetin might reduce the production of TNF-α and IL-1β induced byLPS in the rat brain, as well as simultaneously inhibit the activation of NF-κB signal pathway induced byLPS, with the ultimate goal of inhibiting the in�ammatory factors released by activated microglia [62].Another study also con�rmed that crocin signi�cantly reduced the levels of TNF-α and lipid peroxidationin the brain of PD model animals induced by 6-OHDA, and improved motor ability and cognitive function[63]. Noteworthy, reports indicate that the crocus formula ameliorates the blood glucose level of diabeticrats by regulating intestinal �ora [64]. In summary, the anti PD effect of saffron might involve thein�ammatory reaction mediated by microglia, inhibit the aggregation of α-Syn and regulate the intestinal�ora in exerting a regulatory effect on central in�ammation.

In our study, a series of network pharmacology methods including the OB and DL evaluation, targetprediction, pathway identi�cation, PPI network establishment, and molecular docking technology were

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applied to clarify the underlying mechanism of saffron in the treatment of PD. Our analysis providesconvincing evidence that CASP3, IL6, and MAPK8 are the key targets to expend in the treatment of PDwith saffron. Notably, crocin I and crocin II demonstrated strong binding to CASP3, IL6, and MAPK8, thebinding free energy close to or even less than-8.0 kJ/mol, signi�cantly better compared to others. Besides,the auxiliary neuroprotective effects potentially originated from quercetin, kaempferol, isorhamnetin,crocetin, safranal, and microglial due to their binding capacity to CASP3, IL6, and MAPK8. Our previousstudies revealed that Saffron comprised �avonoids and carotenoids, especially rich in crocin I and crocinII [65]. Moreover, pharmacokinetic studies indicated that crocins were absorbed by passive transcellulardiffusion to a high extent within a short time interval over the intestinal barrier, then, penetrated in a quiteslow process in the blood-brain barrier to reach the central nervous system [66]. All the evidence providedin these studies con�rms the conclusion that saffron exhibits a satisfactory development prospectbecause of the potential neuroprotective effects in the treatment of PD. It is worth noting that, moleculardocking is only a method to simulate the binding mode and a�nity of active components and biologicalmacromolecules by computer software from some algorithms. In fact, the interaction mode of the twomolecules in vivo is very complex and affected by many physiological factors. Future research needs toverify the current results by designing in vitro and in vivo experiments, and provide important informationfor PD drug development.

ConclusionIn summary, we used a network pharmacology strategy to predicate the compounds with biologicalactivity from saffron and the possible targets and pathways related to PD. Consequently, there wassu�cient evidence that crocin I and crocin II play a neuroprotective role by interfering with the target ofapoptosis and in�ammation pathway, and the good drug-target relationship implies that saffron extractmay have potential research and development value in the treatment of PD, namely MAPK8, CASP3 andIL6, which have been predicted as the hopeful target of saffron on PD. The network pharmacology wasan effective tool to explore the material basis of TCM and comprehensively reveal the effects of multi-component, multi-target, and multi-path on the treatment of diseases. However, further experimentalveri�cation is essential, an important next step to consider.

AbbreviationsPD: Parkinson's disease; SNpc:substantia nigra pars compacta; VTA: ventral tegmental area; TCM:Traditional Chinese Medicine; OB: oral bioavailability; DL: drug likeness; GO: Gene Ontology; KEGG: KyotoEncyclopedia of Genes and Genomes; PPI: protein–protein interaction CC: closeness centrality; BC:betweenness centrality; α-Syn: alpha-synuclein; CASP3: caspase-3; IL6: interleukin-6; MAPK8: mitogenactivated protein kinase-8.

DeclarationsAcknowledgements

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Not applicable.

Authors’ contributions

WP conceived and designed the research methods. G-JJ performed the network pharmacology analysisand molecular docking veri�cation. Bathaie SZ, J-GJ revised the manuscript. G-JJ,X-Zj, H-SS, C-BQ, Z-SCwrote the paper. All authors were responsible for reviewing data. All authors read and approved the �nalmanuscript.

Funding

This work was supported by the Special Project of International Technology Cooperation of One Belt andOne Road (No.2017C04009) and the key projects of international scienti�c and technological innovationcooperation between governments (No.2017YFE0130100); Natural Science Foundation of Zhejiang(No.Y18H310026) and General research program of Zhejiang Provincial Department of health(No.2018KY653)

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding authoron reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no con�ict of interest.

Authordetails

1College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, China

2Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran14115111, Iran

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Figures

Figure 1

The �owchart of the network pharmacology-based strategy for predicting the mechanisms of saffron onPD

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Figure 2

The number of therapeutic targets of PD collected in different databases. After merged and de-duplicated,a total of 556 compounds were obtained from 4 databases, including 38 from Drugbank, 104 fromGenecards, 466 from CTD and 29 from OMIM.

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Figure 3

Common targets between candidate compounds targets of saffron and therapeutic targets of PD. 52common targets were observed in both candidate compounds targets of saffron and therapeutic targetsof PD.

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Figure 4

Functional classi�cation of target protein. The largest proportion is apoptosis (19.23%), followed byoxidative stress(13.46), in�ammation(11.54%), cholinergic system disorder(7.69%), and toxic proteindamage(7.69%).

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Figure 5

The drug-candidate compounds targets-therapeutic targets-disease network. The network comprised 153edges and 63 nodes including 52 therapeutic targets of PD represented by green circular nodes, and 9compounds of saffron represented by orange triangle nodes.

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Figure 6

GO enrichment analysis of 52 common targets. The top 6 biological processes implicated in thetreatment of PD were indicated in blue, cellular component were indicated in red, and molecular functionwere indicated in green.

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Figure 7

KEGG enrichment analysis of 52 common targets. The color of the bubbles changed from blue to red,indicating that the statistical signi�cance was gradually enhanced and the size of the circle indicated thenumber of genes enriched in the pathway-the larger the circle, the more genes were enriched.

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Figure 8

The speci�c position and function of common targets in the signaling pathway; (A) PI3K/Akt signalingpathway; (B) apoptosis signaling pathway; (C) TNF signaling pathway.

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Figure 9

Protein‐protein interaction (PPI) network; (A) PPI in the three-dimensional chart; (B) degree values of eachprotein; (C) 3D scatter plot of degree value, closeness centrality and betweenness centrality.

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Figure 10

The heatmap of docking score. The redder the heatmap color is, the smaller the docking score is, thestronger the binding ability between compounds and the proteins.

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Figure 11

Dominant conformation of molecular docking between compounds and target proteins. The moleculardocking was performed by the interaction between the hydrogen bonds interactions of crocin and aminoacid residues of MAPK8, CASP3 and IL6, moreover, the binding strength of crocin I to target protein wasstronger than that of crocin II. Speci�cally, the docking sites of crocin I with MAPK8 including Asn51,Gln37, Ala36, Asn156, and Asp151, and the docking sites with IL6 including Cys146, Leu123, Gln135, and

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Glu140. Crocin I binds with the two peptide chains of CASP3 and forms the largest number of hydrogenbonds interactions. This makes its binding strength stronger than IL6 but less than MAPK8.